Enzyme-catalysed reactions of the type ##STR1## play an important part in biological cells and in biotechnical and analytical reactions. Several hundreds of different hydrogenasas are known which selectively catalyse the conversion of different substrates into products. When the substrate is oxidized, the co-enzymes NAD.sup.+ and NADP.sup.+, respectively, are at the same time reduced to NADH and NADPH, respectively. The major part of the hydrogenases make highly specific demands on the co-enzyme, for which reason NAD.sup.+ and NADP.sup.+, respectively, are necessary to bring about a reaction.
The co-enzymes NAD.sup.+ and NADP.sup.+ are very expensive chemicals which are difficult to obtain. The possibilities of regenerating them by reoxidation therefore are of great economical importance. Regeneration may also be necessary for displacing the equilibrium (1) to the right. In this manner, the substrate can be completely converted into a product, which facilitate the isolation or increase the yield of the desired synthesis products. The displacement may also be a prerequisite for the analytical use of the reaction.
Co-enzyme may be regenerated in different ways:
(a) Chemical regeneration PA0 (b) Enzymatic regeneration PA0 (c) Electrochemical regeneration
Oxidation of co-enzymes with retained activity can be effected only with certain specific oxidizing agents. Such compounds are frequently termed mediators. In FIG. 1, compounds I (phenazine methosulphate, PMS), II (phenazine ethosulphate, PES), III (thionine) and IV (1,2-benzoquinone) are examples of known mediators.
Hitherto known mediators have been used to a limited extent because they, too, are expensive and their stability in the solution may be low. Furthermore, difficulties may arise when the mediators on their reaction and decomposition products are to be isolated from the desired product.
Use is made of an auxiliary reaction, for instance the following: ##STR2## Besides the enzyme according to equation (1), the enzyme lactate dehydrogenase must be added which catalyses a reaction in the opposite direction. If pyruvate is added in excess, NAD.sup.+ will be regenerated.
The use of two separate enzymes makes the biotechnical process more complicated. The reagent of the auxiliary system is mixed with the desired products.
The co-enzyme may be regenerated according to the following reactions: EQU NADH.fwdarw.NAD.sup.+ +2e.sup.- H.sup.+ ( 3) EQU NADPH.fwdarw.NADP.sup.+ +2e.sup.- +H.sup.+ ( 4)
Studies of the electrochemical oxidation of NADH have been reported by H. Jaegfeldt in the Journal of Electroanalytical Chemistry, Vol. 110, p. 295 (1980), by J. Moirouz and P. J. Elving, Analytical Chemistry, Vol. 47, p. 1337 (1975). At pH 7.0, the standard electrode potential of the redox pair NADH/NAD.sup.+ is -0.32 V measured against a standard hydrogen electrode (NHE). The standard electrode potential of NADPH/NADP.sup.+ is almost equally high.
Thermodynamically, the major part (99%) of the co-enzyme could be reacted at an electrode potential which is more anodic than the standard potential by 60mV, i.e. at -0.26 V against NHE. In actual practice, an overvoltage of at least one volt is required for platinum electrodes, while graphite electrodes require an overvoltage immediately below one volt (at least 0.7-0.9 V against NHE). The platinum electrodes require careful pretreatment to prevent side reactions and to give enzymatically active NAD.sup.+ (see H. Jaegfeldt, A. Torstensson and G. Johansson, Analytical Chimica Acta, Vol. 97, p. 221 (1978)). Later studies show that NADPH acts in essentially the same manner as NADH during electrochemical oxidation.
The high overvoltage entails considerable disadvantages. Because the electrode is highly oxidizing, substrates, products, enzymes or other components in the test solution may react in an undesired manner. The enzyme may be denaturized by the high potential. Higher contents of co-enzyme cause side reactions at the electrode surface. The character of these reactions has not been investigated, but it is known that they poison the electrode so that the main reaction is decelerated or discontinued. Different reaction products may polymerize into films inactivating the electrode. Unmodified graphitic materials catalyse a decomposition of the co-enzyme (A. Torstensson, G. Johansson, M-O Mansson, P. O. Larsson and K. Mosbach, Analytical Letters, Vol. 13 B (10)).
Many soluble mediators may be reoxidized electrochemically without excessive overvoltages and may, thus, be used in an auxiliary system for electrochemical regeneration.
In addition to the disadvantages already mentioned and caused by the mixing of the mediator with the reaction products, further difficulties frequently arise because the reduced form of the mediator is difficultly soluble and precipitates.